CN105884879A - Chemical synthesis method of insulin analogue - Google Patents
Chemical synthesis method of insulin analogue Download PDFInfo
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- CN105884879A CN105884879A CN201510037398.0A CN201510037398A CN105884879A CN 105884879 A CN105884879 A CN 105884879A CN 201510037398 A CN201510037398 A CN 201510037398A CN 105884879 A CN105884879 A CN 105884879A
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- insulin
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical class N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 73
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- UGOZVNFCFYTPAZ-IOXYNQHNSA-N levemir Chemical compound CCCCCCCCCCCCCC(=O)NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H]1NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=2N=CNC=2)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=2N=CNC=2)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=2C=CC=CC=2)C(C)C)CSSC[C@@H]2NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)CSSC[C@H](NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC2=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H](CSSC1)C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 UGOZVNFCFYTPAZ-IOXYNQHNSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a chemical synthesis method of insulin analogue. The method comprises four steps of fragment synthesis, fragment condensation, reduction renaturation and purification; a chain A and a chain B of the insulin analogue are synthesized in fragment; when a first disulfide bond is oxidized, Cys-Asn in the chain A and the chain B are oxidized together; due to the fact that the price of Cys-Asn dipeptide is low, when intermolecular oxidation is conducted, dipeptide can be greatly excess so as to improve the intermolecular oxidation efficiency; when dipeptide is oxidized in an excess mode, due to the fact that other active groups do not exist, dipeptide can be recycled in a strong reduction way, and therefore the problem that the yield of intermolecular disulfide bond oxidation is low is solved; an insulin precursor of which the chain A and the chain B are together is obtained through a fragment condensation method, the structure of the insulin precursor is similar to that of insulin of which a chain C exists, renaturation is conducted through a glutathione oxidation-reduction system under a certain condition, and insulin with the high yield is obtained.
Description
Technical field
The present invention relates to the chemical synthesis process of a kind of insulin analog.
Background technology
Diabetes are a kind of common multiple metabolic diseases, and insulin is important endocrine hormone, are the specially good effect polypeptide hormone compounds for the treatment of diabetes.And the problems such as blood glucose fluctuation, injection time be dumb easily occurs in insulin human, in order to solve these problems, scientist, by changing the molecule partial structurtes of insulin human, have developed and obtains more preferably action time and the insulin analog of action effect.
The synthetic method of existing insulin analog is based on fermentation method and synthetic method, and fermentation method is former by fermentation insulin synthesis, then through reduction renaturation, become insulin precurosor, with proteolytic cleavage except C chain, become insulin, by the method for chemosynthesis, add modification group；Shortcoming: owing to there being 3 reactivity amino on insulin, so adding modification group when, it is easy to 3 active reaction amino are modified simultaneously, and the yield causing product is low, purity is low.
Synthetic method: the method the earliest of (1) insulin synthesis is the crystalline bovine insulin of Chinese Academy of Sciences's synthesis, conventional method is all by being respectively synthesized the A chain of insulin and B chain, then insulin is formed by the method for reduction renaturation, but owing to the renaturation process of insulin needs C chain to participate in；Shortcoming: under lacking the response situation that C chain participates in, the productivity of renaturation is extremely low, without productive value；(2) follow-up improve synthesis technique, by being respectively adopted 6 cysteine of different blocking group protection insulins, the method then using a deprotection, form 3 disulfide bond；Shortcoming: the method insulin synthesis yield is the lowest, actual production is not had to be worth, it is primarily due to when 3 disulfide bond are aoxidized by chemical method to introduce 3 kinds of deprotection diverse blocking groups of mechanism, blocking group expensive, the product cost produced is high, on the other hand, oxidizing process needs just to complete for 3 times, especially A chain and B chain disulfide bond are cyclized when, owing to being intermolecular cyclization, form AA chain, BB chain, the probability of AB chain is basically identical, cause yield on the low side, although can be by AA chain, BB chain is cyclized after reopening again, but owing to AA chain and BB chain are all macromole, other active and soft modification can be introduced while opened disulfide bond, last synthesis is caused to be lost efficacy.
Summary of the invention
In order to overcome the defect of prior art, it is an object of the invention to provide the chemical synthesis process of the insulin analog that a kind of yield is high, purity is high, be easy to industrialization produces.
The technical scheme is that
A kind of chemical synthesis process of insulin analog, insulin analog includes A chain and B chain, wherein A chain-ordering is GIVEQCCTSICSLYQLENYCN, B chain-ordering is FVNQHLCGSHLVEALYLVCGERGFFYTPK (R) T, and its synthesis step includes fragment synthesis, fragment condensation, reduction renaturation and four steps of purification:
null(1) fragment synthesis: A chain-ordering is divided into CN fragment、GIVEQCCTSICSLYQLENY fragment is respectively synthesized，B chain-ordering is divided into SHLVEALYLVCGERGFFYTPK (R) T fragment、FVNQHLCG fragment is respectively synthesized,CN fragment uses liquid phase synthesis dipeptides Cys-Asn，GIVEQCCTSICSLYQLENY fragment uses Fmoc solid-phase synthesis synthesis protection polypeptide Boc-GIVEQCCTSICSLYQLENY-COOH，SHLVEALYLVCGERGFFYTPK (R) T fragment uses Fmoc-SHLVEALYLVCGERGFFYTPK (R) T that solid-phase synthesis synthesis N end protection side chain is free，FVNQHLCG fragment uses Fomc solid-phase synthesis synthesis full guard polypeptide fragment Boc-FVNQHLCG-COOH；
(2) fragment condensation: sulfydryl free in-SH and dipeptides Cys-Asn free for Fmoc-SHLVEALYLVCGERGFFYTPK (R) T in step (1) becomes intermediate 1 by disulfide bond oxidative condensation; intermediate 1 and Boc-GIVEQCCTSICSLYQLENY-COOH is condensed into intermediate 2 by amido link; intermediate 2 is condensed into intermediate 3 after sloughing Fmoc by deprotection agent again with Boc-FVNQHLCG-COOH, and intermediate 3 is sloughed all of blocking group by TFA and obtained insulin precurosor；
(3) reduction renaturation: insulin analog precursor in step (2) is dissolved in the renaturation solution of glutathion, renaturation 1-5 days, obtains insulin analog crude product；
(4) purification: insulin crude product step (3) obtained, again through HPLC purification, obtains high purity insulin analog.
Preferably, its step (1) comprises the steps of:
(a1) with Boc-Cys (Trt)-OH and Asn liquid phase synthesis dipeptides Cys-Asn；
(b1) provide a resin as solid support, synthesize the Fmoc of full guard with Fmoc solid phase synthesis process
SHLVEALYLVCGERGFFYTPK (R) T, and with TFA cracking, to obtain aminoterminal be Fmoc, Fmoc SHLVEALYLVCGERGFFYTPK (R) T that side chain is free；
(c1) provide a resin as solid support, synthesize the Boc-GIVEQCCTSICSLYQLENY-COOH fragment of full guard with Fmoc solid phase synthesis process；
(d1) provide a resin as solid support, synthesize the Boc-FVNQHLCG-COOH fragment of full guard with FMOC solid phase synthesis process.
Preferably, its step (2) comprises the steps of:
(a2) sulfydryl free in-SH and Cys-Asn free in Fmoc-SHLVEALYLVCGERGFFYTPK (R) T that N end protection side chain is free by oxidizing become disulfide bond; intermediate 1 after oxidative condensation separates out in reactant liquor, the most stand-by；
(b2) intermediate 1 is dissolved in DMF; add the GIVEQCCTSICSLYQLENY-COOH that aminoterminal is blocking group Fmoc or Boc activated through step (c1); react 12 hours; add water precipitation intermediate 2; through purification of silicagel column; add a deprotection agent and slough blocking group Fmoc or Boc, secondarily purified through silicagel column；
(c2) intermediate 2 is dissolved in DMF; add the FVNQHLCG-COOH that aminoterminal is blocking group Fmoc or Boc activated through step (d1); react 12 hours; add water precipitation intermediate 3; it is purified through silicagel column; add TFA and slough all blocking groups of intermediate 3, obtain insulin precurosor.
Preferably, during step (2) oxidative condensation the mol ratio of Fmoc-SHLVEALYLVCGERGFFYTPK (R) T that Cys-Asn and N end protection side chain is free between 5:1 and 500:1.
Beneficial effects of the present invention: use the dipeptides method of excess, together with Cys-Asn with B chain initial oxidation in A chain, owing to Cys-Asn dipeptides is cheap, during intermolecular oxidation, dipeptides can be significantly excessive, to improve cyclisation efficiency, and at the cyclisation dipeptides of excess, owing to not having other active groups, available strong reduction approach reclaims dipeptides, thus solves the problem that intermolecular cyclization yield is low.
The AB chain obtained by fragment condensation approach insulin precurosor the most together, its structure with C chain insulin type in case seemingly, by being similar glutathione reduction refolding method with biosynthesis, obtains the insulin that yield is the highest.
Detailed description of the invention:
Below in conjunction with embodiment, the invention will be further described:
As R=Myr, representing that this insulin analog is insulin detemir, its synthesis step includes fragment synthesis, fragment condensation, reduction renaturation and four steps of purification:
(1) fragment synthesis
(1) A chain-ordering CN fragment synthesis:
Take 46.4 grams of BOC-Cys(trt) it is dissolved in 1 liter of DMF, add 11.5 grams of HOSU, stir 5 minutes, add 21 grams of DCC, react 24 hours, be filtered to remove precipitation, add 10 liters of cold water, separate out product, after filtration drying, be dissolved in 1 liter of DMF；
Take 25 grams of Asn, add 200ml aqueous solution, add triethylamine regulation PH=9, ASN aqueous solution is added in 1 liter of above-mentioned DMF, reacts 24 hours, add water precipitation precipitation, with silica column purification, obtain the BOC-Cys(trt of 44.5 grams)-Asn, by 44.5 grams of BOC-Cys(trt)-Asn adds 1 liter of 50%TFA dichloromethane solution, react 2 hours, obtain 20 grams of dipeptides Cys-Asn with ether sedimentation；
(2) A chain-ordering BOC-GIVEQCCTSICSLYQLENY fragment synthesis:
Take 100 grams of 2-cl Trt resins, with dichloromethane swelling after, add be dissolved in the Fmoc-Tyr(tbu in 1 liter of DMF)-OH138 gram, react 12 hours, with the Piperidine/DMF solution of 1 liter 20%, slough Fmoc, take 300 grams of FMOC-Asn(trt)-OH, 68 grams of HOBT, 161 grams of TBTU, be dissolved in 1 liter of DMF, add resin, react 4 hours, with the Piperidine/DMF solution of 1 liter 20%, slough fmoc；nullSuccessively by Fmoc-Glu (otbu)-OH、Fmoc-Leu-OH、Fmoc-Gln(trt)-OH、Fmoc-Tyr(tbu)-OH、Fmoc-Leu-OH、Fmoc-Ser(tbu)-OH、Fmoc-Cys(trt)-OH、Fmoc-Ile-OH、 Fmoc-Ser(tbu)-OH、Fmoc-Thr(tbu)-OH、Fmoc-Cys(trt)-OH、Fmoc-Cys(trt)-OH、Fmoc-Gln(trt)-OH、Fmoc-Glu(otbu)-OH、 Fmoc-Val-OH、On the resin of Fmoc-Ile-OH and Fmoc-Gly-OH condensation，After sloughing Fmoc，It is dissolved in 1 liter of DMF with 110 grams of BOC2O，Add resin，React 24 hours；By resin after drying with the dichloromethane solution of 5 liters of 3%TFA, react 1 hour, obtain the polypeptide fragment BOC-GIVEQCCTSICSLYQLENY of 143 grams of full guard；
(3) B chain-ordering SHLVEALYLVCGERGFFYTPK (Myr) T fragment synthesis:
Take 100 grams of wang resins, with dichloromethane swelling after, add be dissolved in the Fmoc-Thr(tbu in 1 liter of DMF)-OH 200 grams, TBTU161 gram, react 12 hours, with the Piperidine/DMF solution of 1 liter 20%, slough Fmoc；Take 243 grams of Fmoc-Lys(Myr)-OH, 68 grams of HOBT, 161 grams of TBTU, be dissolved in 1 liter of DMF, add resin, with the Piperidine/DMF solution of 1 liter 20% after reacting 4 hours, slough Fmoc；nullSuccessively by Fmoc-Pro-OH、Fmoc-Thr(tbu)-OH、Fmoc-Tyr(tbu)-OH、Fmoc-Phe-OH、Fmoc-Phe-OH、Fmoc-Gly-OH、Fmoc-Arg(pbf)-OH、Fmoc-Glu(otbu)-OH、Fmoc-Gly-OH、Fmoc-Cys(trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Tyr(tbu)-OH、Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Glu(otbu)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、On the resin of Fmoc-His (trt)-OH and Fmoc-Ser (tbu)-OH condensation，Retain Fmoc group；By resin after drying by 5 liters of TFA cracked solution, react 2 hours, after ether sedimentation, purify with HPLC, obtain side chain free polypeptide fragment FMOC-SHLVEALYLVCGERGFFYTPK (Myr) T47 gram；
(4) B chain-ordering BOC-FVNQHLCG fragment synthesis:
Take 100 grams of 2-cl Trt resins, with dichloromethane swelling after, add be dissolved in FMOC-Gly-OH89 gram in 1 liter of DMF, react 12 hours, with the Piperidine/DMF solution of 1 liter 20%, slough Fmoc；Take 293 grams of Fmoc-Cys(trt)-OH, 68 grams of HOBT, 161 grams of TBTU, be dissolved in 1 liter of DMF, add resin, with the Piperidine/DMF solution of 1 liter 20% after reacting 4 hours, slough Fmoc；Successively by Fmoc-Leu-OH, Fmoc-His (trt)-OH, Fmoc-Gln (trt)-OH, Fmoc-Asn(trt)-OH, Fmoc-Val-OH and Fmoc-Phe-OH condensation resin on, after sloughing Fmoc, it is dissolved in 1 liter of DMF with 110 grams of BOC2O, add resin, react 24 hours；By resin after drying with the dichloromethane solution of 5 liters of 3%TFA, react 1 hour, obtain the polypeptide fragment BOC-FVNQHLCG of 53 grams of full guard；
(2) fragment condensation
(1) CN Yu FMOC-SHLVEALYLVCGERGFFYTPK (Myr) T
Intermolecular disulfide bond aoxidizes, weigh 20 grams of dipeptides CN, 10 grams of FMOC-SHLVEALYLVCGERGFFYTPK (Myr) T, it is dissolved in 100mlDMF, add 30mlDMSO, react to without free FMOC-SHLVEALYLVCGERGFFYTPK (Myr) T, filter precipitation, obtain 11.6 grams of intermediate 1 after drying；
(2) full guard fragment BOC-GIVEQCCTSICSLYQLENY-COOH is taken
9 grams, it is dissolved in 500ml DMF, adds DCC1 gram; HOSU0.9 gram, react 2 hours, be filtered to remove precipitation; the full guard fragment of activation will be dried to obtain after supernatant water precipitation, 10 grams of intermediate 1 are dissolved in 200mlDMF, add triethylamine regulation PH=9; add the full guard fragment activated; react 24 hours, be dried after aqueous precipitation, slough FMOC with the piperidines of 20%; purify with HPLC, obtain 15 grams of intermediate 2；
(3) take full guard fragment BOC-FVNQHLCG-COOH 6 grams, be dissolved in 500ml DMF, add 1 gram of DCC, 0.9 gram of HOSU; react 2 and be as a child filtered to remove precipitation, the full guard fragment of activation will be dried to obtain after supernatant water precipitation; 15 grams of intermediate 2 are dissolved in 200mlDMF; add triethylamine regulation PH=9, add the full guard fragment activated, react 24 hours; it is dried after aqueous precipitation; shear liquid cracking with TFA, purify with HPLC, obtain 5.8 grams of insulin analog precursor；
(3) reduction renaturation
5.8 grams of insulin analog precursor are dissolved in the glutathion renaturation solution of 1 liter, detected renaturation performance every 12 hours, until renaturation completes；
(4) purification
Insulin analog renaturation completed, by high performance liquid chromatography, is purified, and obtains 3.6 grams of highly purified insulin analog-insulin detemirs, and purity is 98.3%.
Claims (4)
1. the chemical synthesis process of an insulin analog, described insulin analog includes A chain and B chain, wherein A chain-ordering be GIVEQCCTSICSLYQLENYCN, B chain-ordering be FVNQHLCGSHLVEALYLVCGERGFFYTPK (R) T, R is that fatty acid and the like includes but not limited to (C8, C10, C12, C14, C16, C18), R1-Glu (OH)-OH, it is characterised in that: synthesis step includes fragment synthesis, fragment condensation, reduction renaturation and four steps of purification:
null(1) fragment synthesis: A chain-ordering is divided into CN fragment、GIVEQCCTSICSLYQLENY fragment is respectively synthesized，B chain-ordering is divided into SHLVEALYLVCGERGFFYTPK (R) T fragment、FVNQHLCG fragment is respectively synthesized,Described CN fragment uses liquid phase synthesis dipeptides Cys-Asn，Described GIVEQCCTSICSLYQLENY fragment uses Fmoc solid-phase synthesis synthesis full guard polypeptide Boc-GIVEQCCTSICSLYQLENY-COOH，Described SHLVEALYLVCGERGFFYTPK (R) T fragment uses Fmoc-SHLVEALYLVCGERGFFYTPK (R) T that solid-phase synthesis synthesis N end Fmoc protection side chain is free，Described FVNQHLCG fragment uses Fomc solid-phase synthesis synthesis full guard polypeptide fragment Boc-FVNQHLCG-COOH；
(2) fragment condensation: sulfydryl free in-SH and dipeptides Cys-Asn free for Fmoc-SHLVEALYLVCGERGFFYTPK (R) T in step (1) becomes intermediate 1 by disulfide bond oxidative condensation; described intermediate 1 and Boc-GIVEQCCTSICSLYQLENY-COOH is condensed into intermediate 2 by amido link; described intermediate 2 is condensed into intermediate 3 after sloughing Fmoc by deprotection agent again with Boc-FVNQHLCG-COOH, and described intermediate 3 is sloughed all of blocking group by TFA and obtained insulin analog precursor；
(3) reduction renaturation: insulin analog precursor in step (2) is dissolved in the renaturation solution of glutathion, renaturation 1-5 days, obtains insulin analog crude product；
(4) purification: insulin crude product step (3) obtained, again through HPLC purification, obtains high purity insulin analog.
The chemical synthesis process of a kind of insulin analog the most according to claim 1, it is characterised in that: step (1) comprises the steps of:
(a1) with Boc-Cys (Trt)-OH and Asn liquid phase synthesis dipeptides Cys-Asn；
(b1) providing a resin as solid support, with Fmoc SHLVEALYLVCGERGFFYTPK (R) T of Fmoc solid phase synthesis process synthesis full guard, and to obtain aminoterminal with TFA cracking be Fmoc, the Fmoc that side chain is free
SHLVEALYLVCGERGFFYTPK(R)T；
(c1) provide a resin as solid support, synthesize the Boc-GIVEQCCTSICSLYQLENY-COOH fragment of full guard with Fmoc solid phase synthesis process；
(d1) provide a resin as solid support, synthesize the Boc-FVNQHLCG-COOH fragment of full guard with FMOC solid phase synthesis process.
The chemical synthesis process of a kind of insulin analog the most according to claim 1 and 2, it is characterised in that: step (2) comprises the steps of:
(a2) sulfydryl free in-SH and Cys-Asn free in Fmoc-SHLVEALYLVCGERGFFYTPK (R) T that N end protection side chain is free by oxidizing become disulfide bond; intermediate 1 after oxidative condensation separates out in reactant liquor, the most stand-by；
(b2) intermediate 1 that step (a2) obtains is dissolved in DMF; add the GIVEQCCTSICSLYQLENY-COOH that aminoterminal is blocking group Fmoc or Boc activated through step (c1); react 12 hours; add water precipitation intermediate 2; through purification of silicagel column; add a deprotection agent and slough blocking group Fmoc or Boc, secondarily purified through silicagel column；
(c2) step (b2) is obtained intermediate 2 to be dissolved in DMF; add the FVNQHLCG-COOH that aminoterminal is blocking group Fmoc or Boc activated through step (d1); react 12 hours; add water precipitation intermediate 3; it is purified through silicagel column; add TFA and slough all blocking groups of described intermediate 3, obtain insulin precurosor.
The chemical synthesis process of a kind of insulin analog the most according to claim 3, it is characterised in that: during described step (2) oxidative condensation, the mol ratio of Fmoc-SHLVEALYLVCGERGFFYTPK (R) T that Cys-Asn and N end protection side chain is free is between 5:1 and 500:1.
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| CN106432472A (en) * | 2016-10-24 | 2017-02-22 | 合肥国肽生物科技有限公司 | Solid-phase synthesis method for insulin |
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| CN106432472A (en) * | 2016-10-24 | 2017-02-22 | 合肥国肽生物科技有限公司 | Solid-phase synthesis method for insulin |
| CN106432472B (en) * | 2016-10-24 | 2020-01-03 | 合肥国肽生物科技有限公司 | Solid-phase synthesis method of insulin |
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